The present invention relates generally to the development of hypoglycemic effects in mammals, including humans and, more specifically, to novel methods and materials useful in potentiating effects of exogenous insulin on glucose metabolism through use of a synthetic pentadecapeptide which is structurally related to a region in human growth hormone.
The diabetes mellitus disease state is a chronic disorder affecting carbohydrate, fat and protein metabolism. A characteristic feature of idiopathic diabetes mellitus is a defective or deficient insulin secretory response giving rise to impaired carbohydrate (glucose) use and resulting hyperglycemia. Two major variants of the disease state exist. One variant, seen in about ten percent of all idiopathic diabetics, is referred to as insulin-dependent diabetes mellitus ("IDDM") or juvenile onset diabetes. This variant is frequently manifested for the first time in youth and is characterized by a progressive loss of insulin secretory function by beta cells of the pancreas and hence a progressive "dependence" on exogenous insulin for maintenance of carbohydrate metabolism. (This characteristic is shared by those non-idiopathic, or "secondary", diabetics whose disorders have their origins in pancreatic disease.) The second variant of idiopathic diabetes mellitus is referred to as non-insulin-dependent diabetes mellitus ("NIDDM") or adult onset diabetes and accounts for the remainder of the idiopathic diabetic population.
All diabetics, regardless of their genetic and environmental backgrounds or the age of onset of the disease, have in common an apparent lack of insulin or inadequate insulin function. Because transfer of glucose from the blood into muscle and fatty tissue is insulin dependent, diabetics lack the ability to utilize glucose adequately. Further, because glycogenolysis is ordinarily inhibited by insulin, the rate of glycogenolysis is elevated in the diabetic. Both these "derangements" from normal metabolic events lead to accumulation of glucose in the blood (hyperglycemia) to the point where renal glucose reabsorption capacity is exceeded and glycosuria occurs. The major source of energy for the diabetic thus becomes fatty acids derived from triglycerides stored in fatty tissue. In the liver, fatty acids are oxidized to ketone bodies which are circulated and used as an energy source by tissues. In the IDDM patient, and sometimes the NIDDM patient, the rate of formation of ketone bodies may exceed the rate of their utilization and ketosis along with metabolic acidosis may occur. Since tissues appear to be starving for glucose, dietary and tissue sources of protein are used in gluconeogenesis. Anabolic processes such as synthesis of glycogen, triglycerides and proteins are "sacrificed" to catabolic activities including glycogenolysis, gluconeogenesis and mobilization of fats. Thus, the diabetic state which has its origins as a "simple" insulin defect, results in widespread metabolic disturbances having long-term pathologic effects on nearly all organs and tissues of the body. Indeed, the diabetic state is one of the prime contributors to deaths caused by myocardial infarction, renal failure, cerebrovascular disease, atherosclerotic heart disease and systemic infections.
Diabetic therapy for IDDM patients and advanced NIDDM patients has consistently focused on administration of exogenous insulin derived from bovine and porcine sources. It is frequently the case that use of such heterologous species material gives rise to formation of anti-insulin antibodies which have activity-limiting effects and result in progressive requirements for larger doses in order to achieve desired hypoglycemic effects. This, combined with the generally progressive need of the IDDM patient for more exogenous insulin as beta-cell function is lost, tends to accelerate the pathologic effects of the diabetic state.
Use of the most common (and convenient) administrative route for exogenous insulin may itself exacerbate pathology resulting from insulin therapy. Subcutaneous injection of insulin gives rise to relatively high insulin levels in peripheral tissues and relatively low levels circulating through the liver, the primary site of endogenous insulin activity. High levels of insulin in peripheral tissue have been associated with blood vessel pathology (e.g., blood vessel constriction and permeability changes) and pathologic effects on associated peripheral tissues, e.g., diabetic retinopathy. The "swamping" effects of subcutaneously administered insulin on peripheral circulatory tissues eventually reduces the amount of insulin circulating to the liver--again resulting in the need for increased doses to achieve desired metabolic effects.
It will be apparent from the above that substantial long term benefits in insulin therapy for diabetics (especially IDDM patients) can be expected to attend the development of methods and materials for enhancing the hypoglycemic effects of exogenous insulin. If insulin therapy for a given patient is expected to continue over a period of decades, it is significant that initial doses be as small as possible and that large doses of exogenous insulin be avoided for as long as possible.
The recent past has seen modest advances in the development of chemical agents capable of stimulating endogenous insulin secretion and hence reducing the need for exogenous insulin in large doses. Further, recombinant DNA methods have been brought to bear on the problem of securing large scale production of homologous species (human) insulin with the hope that use of the "human" material will reduce the progressive need for larger doses of insulin resulting from the effects of anti-insulin antibodies made against heterologous species materials. As yet, however, no significant advances have been reported in research directed toward development of compounds which would function to augment hypoglycemic effects of any given dose of endogenous insulin and thus guarantee that the insulin dose regimen employed can always be set at or near the minimum needed for desired metabolic effect and will result in the minimum of adverse side effects. There continues to exist, therefore, a need in the art for methods and materials for enhancing the hypoglycemic effects of exogenous insulin in mammals, including humans.
Of interest to the background of the invention are the results of certain studies on insulin-like activities of human growth hormone ("hGH"). hGH is a relatively high molecular weight polypeptide (.sup..about. 22,000 Daltons) consisting of a continuous sequence of 191 amino acid residues with secondary structure provided by two disulfide bonds formed between cysteine residues at position numbers 53/165 and 182/189, respectively. ["Atlas of Protein Sequence and Structure," Vol.5, Supp.2, pp.120-121 (M. Dayhoff, ed., National Biomedical Research Foundation, 1976)]. Early studies of the growth promoting effects of hGH revealed, as one of its intrinsic properties, the ability to initially raise and then lower blood levels of glucose and to lower free fatty acids within one hour of administration, followed by later increasing circulating fatty acids. See, e.g., Goodman, Metabolism, 19, pp. 849-855 (1970); Goodman, Ann. N.Y. Acad. Sci., 148, pp. 419-440 (1968); and Swislocki, et al. Endocrinology, 76, pp. 665-672 (1965). The hyperglycemic and hypoglycemic effects of large doses of hGH are so pronounced in many cases that they constitute a substantial adverse side-effect of hGH therapy for growth disorders.
Determination of the effects of hGH on glycemia prompted a series of studies into the in vivo and in vitro actions of peptide fragments and synthetic fragments related to amino and carboxy terminal regions of hGH. See, e.g., the review by Bornstein appearing at pp.41-49 in "Growth Hormones and Related Peptides," A. Pecile, et al., eds. Excerpta Medica, Amsterdam-Oxford (1976). A variety of biological effects were noted including an insulin potentiating effect on glucose uptake by a fragment duplicating the sequence of amino acid residues at hGH positions 1 through 15 and a hyperglycemic effect for a peptide duplicating residues 176 through 191.
The discovery by Lewis, et al. in 1975 [J.Biol.Chem., 253, pp. 2679-2685] of a naturally-occurring structural variant of hGH which differed from the major form of the hormone by having fewer amino acid residues prompted a systematic examination of the variant, 20,000 Dalton polypeptide, and its properties. Studies by Frigeri, et al., Biochem. Biophys. Res. Comm., 91, pp. 778-782 (1979), Lewis, et al., Biochem. Biophys, Res. Comm., 92, pp. 511-516 (1980), and Lewis, et al., Endocrine Res. Comm., 8, pp. 155-164 (1981) established that the 20,000 Dalton variant lacked the hypoglycemic and fatty acid lowering effects of hGH but substantially retained its growth promotant effects. It was also determined that the "missing" amino acid residues were in a region spanning positions thirty-two to forty-six of hGH. Following these publications were reports of further studies directed toward ascertaining the role of the "missing" residues in the growth stimulating and insulin-like activities of hGH. Frigeri, et al., [Proc. 64th Ann. Meeting of the Endocrine Society, San Francisco, June 1982 (Abstract 88), p. 101]reported that, in normal rats, a synthetic peptide corresponding to residues 32 to 46 of hGH did not show either the late increases in free fatty acids nor the glycemic effects which are characteristic of intact hGH. An unspecified degree of improvement in glucose tolerance of a GT-impaired strain of mice (YS/Wf Nctr) was observed for the peptide, as was an in vitro increase in glucose utilization of insulin-stimulated fat cells of older obese rats. Yudaev, et al., Biochem. Biophys. Res. Comm., 110, pp. 866-872 (1983) reported substantially the same in vitro effects on fat cells for a synthetic tetradecapeptide having a sequence of amino acids copying residues at positions 31 through 44, and reiterated an earlier report of the absence of any in vivo hypoglycemic effect for the tetradecapeptide in rabbits and normal rats. In sum, the above-noted studies revealed that while hGH displays substantial glycemic effects in vivo which are not shown by the 20,000 Dalton variant, the "missing" sequence had no glycemic effect in vivo unless provided to the test animal as part of the hGH polypeptide.